Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis success is critical to the health and fidelityof single cells, multi-cellular development, and disease prevention. In this proposal, we build upon our framework for deciphering the molecular underpinnings of cytokinesis mechanics and mechanosensing. Using Dictyostelium, in Aim 1, we study the Myosin II-Cortexillin I-IQGAP2- Kinesin-6 pathway (the equatorial mechanosensitive pathway). This network of proteins is structured like a mechanochemical feedback system that integrates signals from the mitotic spindle and mechanical stress to tune the myosin II levels at the cleavage furrow. Using fluorescence recovery after photobleaching, we will analyze the dynamics of key proteins with and without applied mechanical stress and in wild type and selected mutants. From this, we will decipher how proteins depend on each other for mechanosensitive accumulation. We will use pull-downs followed by LC-MS to identify interacting proteins. The list of interacting proteins wil then be compared to the lists of genetic interacting proteins we have already identified. Preliminary data identify important enzymes involved in post-translational modifications (PTMs), such as propionylation and acetylation. Acetylation of myosin II and other proteins has been implicated in mammalian mitosis and in cardiac contractile system function. Thus, we are interested to see if these PTMs contribute to the mechanosensory feedback system and cytokinesis cell shape change. We will also use purified proteins to determine how IQGAPs modulate cortexillin and possibly myosin II function. In Aim 2, we will expand the Microtubule-RacE-14-3-3-Myosin II pathway, which we discovered. This pathway controls the global/polar cortex mechanics, cortical tension and cytokinesis shape change. We will draw upon genetic and biochemical methods to identify interactors of racE. We will also determine the mechanism by which 14-3-3 controls myosin II bipolar thick filament assembly. We will then determine how human 14-3-3 proteins modulate human myosin II thick filament assembly. Preliminary data points toward the conserved nature of 14-3-3-myosin II interactions and a possible similar mechanism shared between 14-3-3 and another cancer-related protein S100A4/Mts1. Overall, proposed work in this renewal application strives to develop a sophisticated understanding of the force transmission that promotes and regulates cell shape change and the pathways that control cortical tension, myosin II dynamics, and cytokinesis.